Stilbenedisulfonic Acid Research Articles

Urate Transport via Paracellular Route across Epithelial Cells.

Urate is the final oxidation product of purine metabolism in humans. We have recently reported that the paracellular route is the major urate transport pathway across the blood-placental barrier. In this study, the mechanism of urate paracellular transport was investigated in several epithelial cell lines including Madin-Darby canine kidney (MDCK) type I, Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) and Caco-2 cells. Very little urate passed through MDCK and LLC-PK1 cell layers. In contrast, one of the Caco-2 cell lines was found to be urate-permeable. This urate paracellular movement across Caco-2 cell layer was not inhibited by the urate transporter inhibitor benzbromarone but was partially inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride transport. Detection and quantification of claudin proteins that are important for paracellular transport of ions were performed by LC/MS. Claudins 1, 3, 4, 6, 7 and 12 were detected in urate-permeable cell lines, BeWo cells and Caco-2 cells. We compared claudin expression patterns in urate-permeable and urate-non-permeable Caco-2 cells by LC/MS and found that claudin 12 had a higher expression level in urate-permeable Caco-2 cells. Overexpression of these claudins in MDCK cells did not increase urate paracellular transport. Although there were differences in claudin expression pattern between urate-permeable and non-permeable cells, increased expression of single claudin alone did not explain paracellular permeability of urate.

Properties of single-channel and whole cell Cl- currents in guinea pig detrusor smooth muscle cells.

Multiple types of Cl- channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl- channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl- channels in freshly isolated guinea pig DSM cells. The recorded single Cl- channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of -41.5 mV, which is close to the ECl of -65 mV, confirming preferential permeability to Cl-. The Cl- channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~-20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl- with I-, Br-, or methanesulfonate (MsO-), resulting in anionic permeability sequence: Cl->Br->I->MsO-. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl- current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl- channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

Targeting Anion Exchange of Osteoclast, a New Strategy for Preventing Wear Particles Induced- Osteolysis.

Joint replacement is essential for the treatment of serious joint disease. However, prosthetic failure remains an important clinical issue, with periprosthesis osteolysis (PO), caused by osteoclastic bone resorption induced by wear particles, being the leading cause of failure. Nuclear factor of activated T cells c1 (NFATc1) appears to play an important role in wear particle-induced osteoclastogenesis, with bicarbonate/chloride exchanger, solute carrier family 4, anion exchanger, member 2, (SLC4A2) being upregulated during osteoclastogenesis in an NFATc1-dependent manner. Anion exchange mediated by SLC4A2 in osteoclasts could affect the bone resorption activity by regulating pHi. This study investigated the role and mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function in vitro. The use of 4, 4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an anion exchange inhibitor, suppressed wear particle-induced PO in vivo. Furthermore, controlled release of DIDS from chitosan microspheres can strengthen the PO therapy effect. Therefore, anion exchange mediated by osteoclastic SLC4A2 may be a potential therapeutic target for the treatment of aseptic loosening of artificial joints.

Carbocisteine stimulated an increase in ciliary bend angle via a decrease in [Cl-]i in mouse airway cilia.

Carbocisteine (CCis), a mucoactive agent, is widely used to improve respiratory diseases. This study demonstrated that CCis increases ciliary bend angle (CBA) by 30% and ciliary beat frequency (CBF) by 10% in mouse airway ciliary cells. These increases were induced by an elevation in intracellular pH (pHi; the pHi pathway) and a decrease in the intracellular Cl- concentration ([Cl-]i; the Cl- pathway) stimulated by CCis. The Cl- pathway, which is independent of CO2/HCO3-, increased CBA by 20%. This pathway activated Cl- release via activation of Cl- channels, leading to a decrease in [Cl-]i, and was inhibited by Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid and CFTR(inh)-172). Under the CO2/HCO3--free condition, the CBA increase stimulated by CCis was mimicked by the Cl--free NO3- solution. The pHi pathway, which depends on CO2/HCO3-, increased CBF and CBA by 10%. This pathway activated HCO3- entry via Na+/HCO3- cotransport (NBC), leading to a pHi elevation, and was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The effects of CCis were not affected by a protein kinase A inhibitor (1μM PKI-A) or Ca2+-free solution. Thus, CCis decreased [Cl-]i via activation of Cl- channels including CFTR, increasing CBA by 20%, and elevated pHi via NBC activation, increasing CBF and CBA by 10%.

Synthesis and Characterization of Highly Proton Conducting Sulfonated Polytriazoles

This article describes the synthesis and characterization of highly sulfonated polytriazole copolymers (PTSQSH-I to IV) with IECw values ranging from 2.41 to 3.49 mequiv g–1. The copolymers were synthesized by click reaction between equimolar amount of a dialkyne monomer, potassium 2,5-bis(2-propyn-1-yloxy)benzenesulfonate, and a mixture of two different diazide monomers, 4,4-bis[3′-trifluoromethyl-4′(4-azidobenzoxy)benzyl]biphenyl and 4,4′-diazido-2,2′-stilbene disulfonic acid disodium salt. The copolymers were characterized by Fourier transform infrared and NMR spectroscopy techniques. The membranes were prepared by dissolving the salt form of the copolymers in dimethyl sulfoxide. The copolymers showed high thermal, mechanical, and oxidative stabilities, and the acidified membranes showed very high proton conductivity (43–173 and 132–304 mS cm–1 at 30 and 80 °C, respectively). Transmission electron microscopy images confirmed the formation of well-phase-separated morphology with interconnected hydrophilic domains (20–150 nm).

Attenuation of endothelial phosphatidylserine exposure decreases ischemia-reperfusion induced changes in microvascular permeability.

Translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the endothelial membrane via phospholipid scramblase-1 (PLSCR1) is an apoptotic signal responsible for the loss of endothelial barrier integrity after ischemia-reperfusion injury (IRI). We hypothesized that inhibiting phosphatidylserine expression on endothelial cells would attenuate IRI induced increases in hydraulic permeability (Lp). Mesenteric Lp was measured in rat post-capillary mesenteric venules subjected to IRI via superior mesenteric artery (SMA) occlusion (45 minutes) and release (300 minutes) in conjunction with several inhibitors of phosphatidylserine exposure as follows: (1) inhibition of PLSCR1 translocation (dithioerythritol, n = 3), (2) inhibition of PLSCR1 membrane trafficking (2-bromopalmitate [2-BP], n = 3), and (3) inhibition of ion exchange necessary for PLSCR1 function (4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid [DIDS], n = 3). Under the same IRI conditions, rats were also administered targeted inhibitors of phosphatidylserine exposure including knockdown of PLSCR1 (n = 3) using RNA interference (RNAi), and as a potential therapeutic tool Diannexin, a selective phosphatidylserine blocker (n = 3). During IRI net Lp increased by 80% (p < 0.01). Net reductions of Lp were accomplished by 2-BP (46% reduction, p = 0.005), combined DET + 2-BP + DIDS (32% reduction, p = 0.04), RNAi (55% reduction, p = 0.002), Diannexin administered pre-SMA artery occlusion (73% reduction, p = 0.001), and post-SMA occlusion (70% reduction, p = 0.002). Phosphatidylserine exposure is a key event in the pathogenesis of microvascular dysfunction during IRI. Clinically, inhibition of phosphatidylserine exposure is a promising strategy that may 1 day be used to mitigate the effects of IRI.

New crosslinked sulfonated polytriazoles: Proton exchange properties and microbial fuel cell performance

The synthesis, characterization and establishment of the structure-property relationship of a series of new semifluorinated sulfonated polytriazoles (PTCTSH-XX) with varying degrees of sulfonation are described. The polymers were synthesized by the click polymerization reaction of a mixture of two diazide monomers, the non-sulfonated diazide monomer 4,4-bis[3-trifluoromethyl-4(4-azidophenoxy) phenyl]benzene (TAZ) and the sulfonated diazide monomer 4,4′-diazido-2,2′-stilbenedisulfonic acid disodium salt (SAZ), with an equimolar amount of the dialkyne monomer 3,5-bis(prop-2-ynyloxy)benzoic acid (BPBA). The polytriazoles were characterized by 1H and 13C NMR spectroscopy. All the polymers showed good solubility in selected polar aprotic organic solvents along with excellent film forming ability. The TEM micrographs of these polymers exhibited a uniformly phase separated morphology. The polymers showed very high proton conductivity and the values were within the range of 15–67 mS cm−1 at 30 °C, 38–163 mS cm−1 at 80 °C and 42–176 mS cm−1 at 90 °C in hydrated condition. The pristine polymers were crosslinked with poly(vinyl alcohol) and their PEM properties were also investigated. The crosslinked films showed lower water uptake and dimensional swelling and improved oxidative stability in comparison to the pristine membranes. The microbial fuel cell performance of both pristine and crosslinked membranes was also investigated.

Comb-shaped polysulfones containing sulfonated polytriazole side chains for proton exchange membranes

Comb-shaped polysulfone copolymers were synthesized by the click reaction, copper(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition, using polysulfone having azidomethyl side group (PSf-N3) and sulfonated polytriazole having one ethynyl chain-end group. PSf-N3 was prepared by the substitution reaction of polysulfone through chloromethylation followed by azidation, and sulfonated polytriazoles having one ethynyl chain-end group was synthesized via the click reaction using 1,4-diethynylbenzene and 4,4′-diazido-2,2′-stilbenedisulfonic acid disodium salt tetrahydrate with CuI followed by end-capping process. Tough, flexible, and transparent membranes could be prepared by solution casting from the comb-shaped polysulfone copolymers and ion exchange capacity (IEC) of the resulting membranes could be controlled by changing the chain length of the sulfonated polytriazoles. The comb-shaped polysulfone membranes showed enhanced physicochemical stability and significantly higher proton conductivity when compared with a sulfonated poly(arylene ether sulfone) (SPAES) membrane having a similar IEC due to the distinct hydrophilic-hydrophobic phase separated morphology. The fuel cell performance of membrane electrode assembly with the comb-shaped polysulfone membrane is superior to those with the SPAES and Nafion-212 membranes at the various operating conditions.

Sequential alkyne-azide cycloadditions for functionalized gelatin hydrogel formation

While click chemistry reactions for biopolymer network formation are attractive as the defined reactions may allow good control of the network formation and enable subsequent functionalization, tailoring of gelatin network properties over a wide range of mechanical properties has yet to be shown. Here, it is demonstrated that copper-catalyzed alkyne-azide cycloaddition of alkyne functionalized gelatin with diazides gave hydrogel networks with properties tailorable by the ratio of diazide to gelatin and diazide rigidity. 4,4′-diazido-2,2′-stilbenedisulfonic acid, which has been used as rigid crosslinker, yielded hydrogels with Young’s moduli E of 50–390 kPa and swelling degrees Q of 150–250 vol.%, while the more flexible 1,8-diazidooctane resulted in hydrogels with E = 125–280 kPa and Q = 225–470 vol.%. Storage moduli could be varied by two orders of magnitude (G′ = 100–20,000 Pa). An indirect cytotoxicity test did not show cytotoxic properties. Even when employing 1:1 ratios of alkyne and azide moieties, the hydrogels were shown to contain both, unreacted alkyne groups on the gelatin backbone as well as dangling chains carrying azide groups as shown by reaction with functionalized fluorescein. The free groups, which can be tailored by the employed ratio of the reactants, are accessible for covalent attachment of drugs, as was demonstrated by functionalization with dexamethasone. The sequential network formation and functionalization with click chemistry allows access to multifunctional materials relevant for medical applications.

Permeation of acetate across sheep ruminal epithelium is partly mediated by an anion channel

The present study pursued the question if an anion channel could be partly responsible for the transport of acetate in the isolated ovine ruminal epithelium. Using the Ussing chamber technique, changes of short-circuit current (Isc) induced by mucosal or serosal addition of acetate was investigated. To further evaluate the Isc changes induced by acetate the epithelia were preincubated with ouabain or 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). In addition, unidirectional flux rates of 14C-acetate were measured at different transepithelial potential difference (Vt). At Vt=0mV, acetate addition to the mucosal side of epithelia incubated in ouabain/DIDS free solution resulted in an Isc decrease, whereas application to the serosal side induced an Isc increase. Absolute Isc changes were significantly larger after serosal than after mucosal acetate addition. Ouabain pre-incubation abolished these side-specific differences. Pre-incubation with DIDS on the mucosal side inhibited the current induced by subsequent mucosal acetate addition in a voltage dependent manner, whereas serosal DIDS pre-incubation had no effect on acetate-induced changes of Isc. Mucosal-to-serosal acetate flux was partly voltage-dependent. The serosal-to-mucosal flux of acetate was not influenced by Vt. The asymmetric changes of Isc after acetate application and the Vt dependence of the DIDS inhibition on the mucosal side indicate a partly electrogenic transcellular permeation of acetate which includes an apically located acetate conductance. It is suggested that this conductance may be important for epithelial cell homeostasis.

1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture

The major circulating metabolite of vitamin D3, 25-hydroxycholecalciferol [25(OH)D], has a remarkably long half-life in blood for a (seco)steroid. Data from our studies and others are consistent with the hypothesis that there is a role for skeletal muscle in the maintenance of vitamin D status. Muscle cells internalise vitamin D-binding protein (DBP) from the circulation by means of a megalin/cubilin plasma membrane transport mechanism. The internalised DBP molecules then bind to actin and thus provide an intracellular array of high affinity binding sites for its specific ligand, 25(OH)D. There is evidence that the residence time for DBP in muscle cells is short and that it undergoes proteolytic degradation, releasing bound 25(OH)D. The processes of internalisation of DBP and its intracellular residence time, bound to actin, appear to be regulated. To explore whether 1,25-dihydroxycholecalciferol (calcitriol) has any effect on this process, cell cultures of myotubes and primary skeletal muscle fibers were incubated in a medium containing 10−10M calcitriol but with no added DBP. After 3h pre-incubation with calcitriol, the net uptake of 25(OH)D by these calcitriol-treated cells over a further 4h was significantly greater than that in vehicle-treated control cells. This was accompanied by a significant increase in intracellular DBP protein. However, after 16h of pre-incubation with calcitriol, the muscle cells showed a significantly depressed ability to accumulate 25(OH)D compared to control cells over a further 4 or 16hours. These effects of pre-incubation with calcitriol were abolished in fibers from VDR-knockout mice. The effect was also abolished by the addition of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride channel opening. Incubation of C2 myotubes with calcitriol also significantly reduced retention of previously accumulated 25(OH)D after 4 or 8h. It is concluded from these in vitro studies that calcitriol can modify the DBP-dependent uptake and release of 25(OH)D by skeletal muscle cells in a manner that suggests some inducible change in the function of these cells.

Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

ATP fuels the removal of metabolic end-products, including H+ ions that profoundly modulate biological activities. Energetic resources in hypoxic tumor regions are constrained by low-yielding glycolysis, and any means of reducing the cost of acid extrusion, without compromising pH homeostasis, would therefore be advantageous for cancer cells. Some cancers express connexin channels that allow solute exchange between cells, and we propose that, via this route, normoxic cells supply hypoxic neighbors with acid-neutralizing HCO3- ions. This hypothesis was tested by imaging cytoplasmic pH in spheroidal tissue growths of connexin43-positive pancreatic cancer Colo357 cells during light-controlled H+ uncaging at the hypoxic core. Cytoplasmic acid retention at the core was halved in the presence of CO2/HCO3-, but this process requires a restorative HCO3- flux. The effect of CO2/HCO3- was ablated by connexin43 inhibition or knockdown. In connexin-decoupled spheroids, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an inhibitor of HCO3- uptake, had no effect on cytoplasmic [H+] in the H+-uncaging region, indicating that DIDS-sensitive transport is not an adequate pH-regulatory strategy therein. With intact connexin-coupling, acid retention at the core increased upon DIDS treatment, indicating that HCO3- ions are taken up actively by peripheral cells and then transmitted passively to cells at the hypoxic core. Thus, the energetic burden of pH regulation is offloaded from hypoxic cells onto metabolically altruistic normoxic neighbors.-Dovmark, T. H., Hulikova, A., Niederer, S. A., Vaughan-Jones, R. D., Swietach, P. Normoxic cells remotely regulate the acid-base balance of cells at the hypoxic core of connexin-coupled tumor growths.

Hydroquinone Based Sulfonated Copolytriazoles with Enhanced Proton Conductivity

AbstractA series of new fluorinated sulfonated copolytriazoles (PTHQSH‐XX) with ion exchange capacity (IECw) values ranging from 1.66 to 2.82 meq g−1 are prepared via cuprous ion catalyzed azide‐alkyne click polymerization reaction between 1,4‐bis(prop‐2‐ynyloxy)benzene, 4,4′‐diazido‐2,2′‐stilbene disulfonic acid disodium salt (SA), and 4,4‐bis[3′‐trifluoromethyl‐4′(4‐azidobenzoxy) benzyl] biphenyl (QAZ). The degree of sulfonation of the copolytriazoles is adjusted between 60% and 90% by varying the molar ratio of sulfonated monomer (SA) to the nonsulfonated monomer (QAZ). The structure of the copolytriazoles is characterized by Fourier transform infrared and NMR spectroscopy. The solution‐cast membranes of these copolymers exhibit high thermal, mechanical, oxidative and hydrolytic stability, and high proton conductivity (19–142 mS cm−1 at 80 °C and 22–157 mS cm−1 at 90 °C). Transmission electron microscopy confirms the formation of good phase separated morphology with ionic clusters in the range of 15–145 nm.

Flexible Diazide Based Sulfonated Polytriazoles and Their Proton Exchange Membrane Properties

The preparation, characterization, and property evaluation of a series of new semifluorinated polytriazoles (PTEHSH‐XX) with various degrees of sulfonation is described. The polymers are synthesized by the click polymerization reaction of a mixture of a new diazide monomer, 1,4‐bis‐[{2′‐trifluromethyl 4′‐(4″‐azidophenyl)phenoxy}]phenyl, and a sulfonated monomer, 4,4′‐diazido‐2,2′‐stilbenedisulfonic acid disodium salt, with an equimolar amount of a dialkyne monomer, 1,3‐diethynylbenzene. The polytriazoles are characterized by Fourier transform infrared spectroscopy (FTIR) and NMR spectroscopic techniques. The polytriazoles show good solubility in selective organic solvents and film forming ability. The films prepared from these polymers show high thermal and chemical stability, low water uptake, high dimensional stability, and excellent mechanical properties. The transmission electron microscopy images of these polymers exhibit a uniformly phase separated morphology. The polymers show very high proton conductivity and the values are within the range of 27–118 mS cm−1 at 30 °C, 53–225 mS cm−1 at 80 °C, and 58–235 mS cm−1 at 90 °C in hydrated condition. image

Estradiol activates chloride channels via estrogen receptor-α in the cell membranes of osteoblasts.

Estrogen plays important roles in regulation of bone formation. Cl- channels in the ClC family are expressed in osteoblasts and are associated with bone physiology and pathology, but the relationship between Cl- channels and estrogen is not clear. In this study the action of estrogen on Cl- channels was investigated in the MC3T3-E1 osteoblast cell line. Our results show that 17β-estradiol could activate a current that reversed at a potential close to the Cl- equilibrium potential, with a sequence of anion selectivity of I- > Br- > Cl- > gluconate, and was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid. Knockdown of ClC-3 Cl- channel expression by a specific small interfering RNA to ClC-3 attenuated activation of the 17β-estradiol-induced Cl- current. Extracellular application of membrane-impermeable 17β-estradiol-albumin conjugates activated a similar current. The estrogen-activated Cl- current could be inhibited by the estrogen receptor (ER) antagonist fulvestrant (ICI 182780). The selective ERα agonist, but not ERβ agonist, activated a Cl- current similar to that induced by 17β-estradiol. Silencing ERα expression prevented activation of estrogen-induced currents. Immunofluorescence and coimmunoprecipitation experiments demonstrated that ClC-3 Cl- channels and ERα were colocalized and closely related in cells. Estrogen promoted translocation of ClC-3 and ERα to the cell membrane from the nucleus. In conclusion, our findings show that Cl- channels can be activated by estrogen via ERα on the cell membrane and suggest that the ClC-3 Cl- channel may be one of the targets of estrogen in the regulation of osteoblast activity.

Evolutionarily distinct strategies for the acquisition of inorganic carbon from seawater in marine diatoms.

The acquisition of dissolved inorganic carbon (DIC) in CO2-limited seawater is a central issue to understand in marine primary production. We previously demonstrated the occurrence of direct HCO3- uptake by solute carrier (SLC) 4 transporters in a diatom, a major marine primary producer. Homologs of SLC are found in both centric and pennate marine diatoms, suggesting that SLC transporters are generally conserved. Here, the generality of SLC-mediated DIC uptake in diatoms was examined using an SLC inhibitor, diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and an inhibitor of external carbonic anhydrase, acetazolamide. DIDS suppressed high-DIC-affinity photosynthesis in the pennate diatom Phaeodactylum tricornutum and the centric diatom Chaetoceros muelleri, but there was no effect on either the pennate Cylindrotheca fusiformis or the centric Thalassiosira pseudonana. Interestingly, the DIC affinity of DIDS-insensitive strains was sensitive to treatment with up to 100 μM acetazolamide, displaying a 2-4-fold increase in K0.5[DIC]. In contrast, acetazolamide did not affect the DIDS-sensitive group. These results indicate the occurrence of two distinct strategies for DIC uptake-one primarily facilitated by SLC and the other being passive CO2 entry facilitated by external carbonic anhydrase. The phylogenetic independence of these strategies suggests that environmental demands drove the evolution of distinct DIC uptake mechanisms in diatoms.

CAMP-dependent secretagogues stimulate the NaHCO3 cotransporter in the villous epithelium of the brushtail possum, Trichosurus vulpecula.

In the ileum of the brushtail possum, Trichosurus vulpecula, fluid secretion appears to be driven by electrogenic HCO3- secretion. Consistent with this, the cystic fibrosis transmembrane conductance regulator is expressed in the apical membrane of the ileal epithelial cells and the pancreatic or secretory variant of the NaHCO3 cotransporter in the basolateral membrane. This suggests that in the possum ileum, electrogenic HCO3- secretion is driven by basolateral NaHCO3 cotransporter (NBC) activity. To determine if the NBC contributes to HCO3- secretion in the possum ileum, intracellular pH (pHi) measurements in isolated villi were used to demonstrate NBC activity in the ileal epithelial cells and investigate the effect of cAMP-dependent secretagogues. In CO2/HCO3--free solutions, recovery of the epithelial cells from an acid load was Na+-dependent and ≈80% inhibited by ethyl-isopropyl-amiloride (EIPA, 10µmol L-1), indicative of the presence of an Na+/H+ exchanger, most likely NHE1. However, in the presence of CO2/HCO3-, EIPA only inhibited ≈ 50% of the recovery, the remainder was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 500µmol L-1), indicative of NBC activity. Under steady-state conditions, NHE1 inhibition by EIPA had little effect on pHi in the presence or absence of secretagogues, but NBC inhibition with DIDS resulted in a rapid acidification of the cells, which was increased fivefold by secretagogues. These data demonstrate the functional activity of an NaHCO3 cotransporter in the ileal epithelial cells. Furthermore, the stimulation of NBC activity by secretagogues is consistent with the involvement of an NaHCO3 cotransporter in electrogenic HCO3- secretion.

Hexafluoroisopropylidene based sulfonated new copolytriazoles: investigation of proton exchange membrane properties

Abstract A series of novel sulfonated polytriazole copolymers (PTFOSH-XX) was successfully prepared by the click reaction of 4,4′-(perfluoropropane-2,2-diyl)bis((prop-2-ynyloxy)benzene (TF), 4,4′-diazido-2,2′-stilbene disulfonic acid disodium salt (SAZ) and 4,4′-diazidodiphenyl ether (OAZ). The copolymers were characterized by Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (NMR) spectroscopy. The copolymers showed high mechanical, thermal and oxidative stability and low swelling. The phase separated morphology of the membranes was confirmed from transmission electron microscopy (TEM). The membranes showed proton conductivity as high as 110 and 122 mS cm−1 at 80 and 90°C, respectively depending on the polymer repeat unit structure.

Combined “post-infiltration, subsequent photochemical cross-linking” and “cross-linking and selective etching” strategies to fabricate nanoporous layer-by-layer assembled multilayers

This study reports a new method combining a “post-infiltration, subsequent photochemical cross-linking” strategy and a “cross-linking and selective etching” approach to fabricate porous multilayers incorporating weak polyelectrolytes poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), and poly(sodium-p-styrenesulfonate) (PSS). The multilayer film is fabricated by layer-by-layer (LbL) assembly of a blend of PAH and PEI in alternation with PSS to construct a composite multilayer film as a precursor. Then, 4,4′-diazido-2,2′-stilbenedisulfonic acid disodium (DAS) is infiltrated into the multilayer films, and subsequent photochemical cross-linking is applied under UV irradiation. By taking advantage of the differences between PAH/PSS and PEI/PSS, the multilayer films are immersed into a basic solution to selectively dissolve PEI. UV-visible spectroscopy, atomic force microscopy and cyclic voltammogram characterization results prove formation of nanoporous structures in the LbL assembled multilayer films. This novel way to fabricate porous films is anticipated to have potential applications in polymer and interface science.

Expression of anoctamins in retinal pigment epithelium (RPE).

The anoctamin (ANO, TMEM16) family of Ca2+-activated Cl- channels consists of ten members with different cellular functions (ANO1-10). ANO1 is a Ca2+-activated Cl- channel in secretory epithelial cells of exocrine pancreas, salivary glands, or enterocytes. Expression of ANO1 also promotes cell proliferation and migration of tumor cells. ANO6 is essential for Ca2+-dependent scrambling of membrane phospholipids in platelets, red blood cells, and lymphocytes. ANO10 modulates Ca2+ signals in macrophages and has a role in cerebellar ataxia and other neurological disorders. All three anoctamins have been proposed to control intracellular Ca2+ signals. Anoctamins may also form the basolateral Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE). We show that native human, bovine, porcine, and mouse RPEs express ANO1, ANO6, and ANO10. Growth arrested and confluent RPR cells expressed ANO1 in the plasma membrane, whereas ANO6 and ANO10 were found in the primary cilium. Ussing chamber experiments showed that the application of ATP to the apical (retinal) side of porcine RPE induced a Ca2+-activated Cl- secretion. Activation was inhibited by basolateral (choroidal) administration of the ANO inhibitors AO1, niflumic acid (NFA), and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). The results suggest that ANO1 is responsible for basolateral Ca2+-dependent Cl- secretion in RPE, whereas ANO6 and ANO10 may have different functions, such as modulating Ca2+ signals.